Within the scope of the present invention, a microcarrier or a microparticle refers to any type of particles, respectively to any type of carriers, microscopic in size, typically with the largest dimension being from 100 nm to 300 μm, preferably from 1 μm to 200 μm.
According to the present invention, the term microcarrier refers to a microparticle functionalized, or adapted to be functionalized, that is containing, or adapted to contain, one or more ligands or functional units bound to the surface of the microcarrier or impregnated in its bulk. A large spectrum of chemical and biological molecules may be attached as ligands to a microcarrier. A microcarrier can have multiple functions and/or ligands. As used herein, the term functional unit is meant to define any species that modifies, attaches to, appends from, coats or is covalently or non-covalently bound to the surface of said microcarrier or impregnated in its bulk. These functions include all functions that are routinely used in high-throughput screening technology and diagnostics.
Drug discovery or screening and DNA sequencing commonly involve performing assays on very large numbers of compounds or molecules. These assays typically include, for instance, screening chemical libraries for compounds of interest or particular target molecules, or testing for chemical and biological interactions of interest between molecules. Those assays often require carrying out thousands of individual chemical and/or biological reactions.
Numerous practical problems arise from the handling of such a large number of individual reactions. The most significant problem is probably the necessity to label and track each individual reaction.
One conventional method of tracking the identity of the reactions is achieved by physically separating each reaction in a microtiter plate (microarray). The use of microtiter plates, however, carries several disadvantages like, in particular, a physical limitation to the size of microtiter plates used, and thus to the number of different reactions that may be carried out on the plates.
In light of the limitations in the use of microarrays, they are nowadays advantageously replaced by functionalized encoded microparticles to perform chemical and/or biological assays. Each functionalized encoded microparticle is provided with a code that uniquely identifies the particular ligand(s) bound to its surface. The use of such functionalized encoded microparticles allows for random processing, which means that thousands of uniquely functionalized encoded microparticles may all be mixed and subjected to an assay simultaneously. Examples of functionalized encoded microparticles are described in the international patent application WO 00/63695 and are illustrated in FIG. 1.
The international patent application WO 2010/072011 describes an assay device having at least a microfluidic channel which serves as a reaction chamber in which a plurality of functionalized encoded microparticles or microcarriers 1 (FIG. 1) can be packed. The microfluidic channel is provided with stopping means acting as filters that allow a liquid solution containing chemical and/or biological reagents to flow through while blocking the microcarriers 1 inside. The geometrical height of said microfluidic channels and the dimensions of said microcarriers 1 are chosen so that said microcarriers 1 are typically arranged in a monolayer arrangement inside each microfluidic channel preventing said microcarriers 1 to overlap each other.
Those functionalized encoded microcarriers 1 that show a favorable reaction of interest between their attached ligand(s) and the chemical and/or biological reagents flowing through may then have their code read, thereby leading to the identity of the ligand that produced the favorable reaction.
The code may comprise a distinctive pattern including a plurality of traversing holes 2 and an asymmetric orientation mark such as, for example, a L-shaped sign 3 (as shown in FIG. 1) or a triangle. This asymmetric orientation mark allows the distinction between the top surface 4 and the bottom surface 5 of the microcarrier 1.
The term microfluidic channel refers to a closed channel, i.e. an elongated passage for fluids, with a cross-section microscopic in size, i.e. with the smallest dimension of the cross-section being typically from about 1 to about 500 micrometers, preferably about 10 to about 200 micrometers. A microfluidic channel has a longitudinal direction, that is not necessarily a straight line, and that corresponds to the direction in which fluids are flowing within the microfluidic channel, i.e. preferably essentially to the direction corresponding to the average speed vector of the fluid, assuming a laminar flow regime.
With the assay device described in WO 2010/072011, the detection of a reaction of interest can be based on continuous readout of the fluorescence intensity of each encoded microcarrier 1 present in a microfluidic channel. In other words, the presence of a target molecule in the assay will trigger a predetermined fluorescent signal.
However, the functionalized encoded microparticles 1 and the assay device described in WO 2010/072011 do not allow for rapid quantification of reagent or ligand before an equilibrium state is reached, when the fluorescent signals saturate. Although the assay device of WO 2010/072011 decreases the time needed to reach equilibrium, in typical concentration values of analyte in the nano-molar range, ten to twenty minutes are still required, while lower concentration in the pico-molar range can take up to hours to be reached and serve for quantification. Moreover, the discrepancies in their fluorescent signals, in particular the diffusion pattern even after the equilibrium has been reached do not determine a quantitative information with a lower margin of error than about 15%.
To remedy these drawbacks, the patent application PCT/CH2012/000032 proposes an encoded microcarrier, shown in FIGS. 2 and 3, comprising a body 6 having a shape of a right circular cylinder and comprising a top surface 4, a bottom surface 5 and spacing elements 7 protruding from the bottom surface 5.
The microcarrier 1 with its spacing elements 7 is shaped to ensure that, when the encoded microcarrier 1 is laid on a flat plane 8 with the detection surface 5 facing said plane 8, a gap d exists between said flat plane 8 and the detection surface 5, as shown in FIG. 3.
As said above, the encoded microcarrier contains one or more ligands bound to the bottom surface 5 (detection surface). When contacting the ligand-bound encoded microcarrier 1 with a solution that may contain one or more target analytes, a reaction of interest may occur on the detection surface 5, depending on the presence or absence of a proper analyte. As an example, a reaction of interest can emit or inhibit a fluorescent signal, which can be monitored. Detecting a reaction on the detection surface 5 can allow determining the presence or absence of particular analytes of interest.
The document PCT/CH2012/000032 also discloses an assay system comprising a plurality of encoded microcarriers 1 with spacing elements and an assay device, partially shown in FIGS. 4 and 5. The assay device 9 has at least one microfluidic channel 10 having an inlet connected to an inlet well 11 and an outlet connected to an outlet well 12, said channel 10 being shaped to accommodate a plurality of said encoded microcarriers 1. The microfluidic channel 10 is provided with stopping means 13 arranged in the vicinity of the outlet of the microfluidic channel 10 and acting as a filter that allow a liquid solution to flow through while blocking said encoded microcarriers 1 inside. The microfluidic channel 10 has a cross-section that allows at least two encoded microcarriers 1 to be arranged side by side over the length of said microfluidic channel 10, in a monolayer arrangement as depicted in FIG. 5. The microfluidic channel 10 comprises at least an observation wall 14 through which an assay is monitorable. Typically, when the assay is monitored by fluorescent signal, the observation wall is transparent.
In such an assay system, when the encoded microcarriers are loaded in the microfluidic channel 10 with said detection surface 5 facing said observation wall 14, the spacing elements 7 generate a gap d between said detection surface 5 and said observation wall 14 to allow a circulation of liquid in said gap d, said liquid containing chemical and/or biological reagent of interest for the assay.
Thus, the spacing elements 7 permit a homogeneous convective flow all over the microfluidic channel 10 resulting in homogeneous fluorescent increase over time and across encoded microcarriers 1. The homogeneous signal increase allows for a rapid quantification of the analyte being flushed, from the first seconds, by monitoring the fluorescence rate.
When microcarriers 1 are introduced into the inlet well 11, said microcarriers 1 may flip over during their sedimentation in the well 11. Thus, some of the microcarriers 1 have their detection surface 5 opposite to the detection wall 14 of the microchannel 10. However, the detection of the presence of molecules bound to the detection surface 5 such as by fluorescence microscopy is possible only when said surface 5 is facing the detection wall 11. Thus, microcarriers 1 having a wrong orientation do not emit any detectable signal.
Moreover, the laminar fluid flow is disturbed by the microcarriers 1 which are not properly oriented. Indeed, in this case, said laminar fluid flow is forced to move around the concerned microcarriers 1, thus creating disturb the homogeneity of the fluid velocity along the microchannel 10, thereby disturbing the homogeneous distribution of the reagents and target molecules intended to interact with the detection surfaces 5 of the microcarriers 1. This affects the reliability of the assay.
More generally, the same orientation problem could arise with other kind of microcarriers having only one of the bottom and top surfaces provided with a three-dimensional structure.